Method for determining the three-dimensional structure of a protein

ABSTRACT

Microcapsules prepared by encapsulating an aqueous solution of a protein, drug or other bioactive substance inside a semi-permeable membrane by are disclosed. The microcapsules are formed by interfacial coacervation under conditions where the shear forces are limited to 0-100 dynes/cm 2  at the interface. By placing the microcapsules in a high osmotic dewatering solution, the protein solution is gradually made saturated and then supersaturated, and the controlled nucleation and crystallization of the protein is achieved. The crystal-filled microcapsules prepared by this method can be conveniently harvested and stored while keeping the encapsulated crystals in essentially pristine condition due to the rugged, protective membrane. Because the membrane components themselves are x-ray transparent, large crystal-containing microcapsules can be individually selected, mounted in x-ray capillary tubes and subjected to high energy x-ray diffraction studies to determine the 3-D structure of the protein molecules. Certain embodiments of the microcapsules of the invention have composite polymeric outer membranes which are somewhat elastic, water insoluble, permeable only to water, salts, and low molecular weight molecules and are structurally stable in fluid shear forces typically encountered in the human vascular system.

This application is a divisional of U.S. Patent Application No.09/079,766 filed May 15, 1998, now U.S. Pat. No. 6,387,399, which is acontinuation-in-part of U.S. Patent Application No. 08/349,169 filedDec. 2, 1994, now U.S. Pat. No. 5,827,531.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The invention described herein was made in the performance of work undera NASA contract and is subject to Public Law 96-517(35 U.S.C. § 200 etseq.). The contractor has not elected to retain title to the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to methods of microencapsulatingbioactive substances, and particularly to methods utilizing interfacialcoacervation at the immiscible interface of two liquid phases. Moreparticularly, the invention pertains to such methods which maintainconditions of low shear force during formation of the microcapsules. Thepresent invention also pertains to microcapsules formed by such methodsand to their methods of use.

2. Description of the Prior Art

Liquid microcapsules and liposomes are often used to store and deliverbioactive substances such as drugs, enzymes or biocatalysts. One recenteffort to provide liposomes with enhanced circulation times is thatdisclosed in U.S. Pat. No. 5,013,556 to Woodle et al. Liposomes createdby Woodle et al. contain 1-20 mole % of an amphipathic lipid derivatizedwith a polyalkylether (such as phosphatidyl ethanolamine derivitizedwith polyethyleneglycol). Another improvement is provided by U.S. Pat.No. 5,225,212 (issued to Martin et al.) which discloses a liposomecomposition for extended release of a therapeutic compound into thebloodstream. Those liposomes are composed of vesicle-forming lipidsderivatized with a hydrophilic polymer, wherein the liposome compositionis used for extending the period of release of a therapeutic compoundsuch as a polypeptide, injected within the body. Formulations of“stealth” liposomes have also been created with lipids that are lessdetectable by immune cells in an attempt to avoid phagocytosis (Allen etal. (1992) Cancer Res. 52:2431-39.) Still other modifications of lipids(i.e., neutral glycolipids) may be made in order to produce anti-viralformulations. U.S. Pat. No. 5,192,551 to Willoughby et al. 1993.However, new types of liposomes and microcapsules are needed to exploitthe various unique applications of this type of drug delivery.

Many proteins of interest, such as those containing bioactive drug sitesor enzymatically active sites, are only slightly soluble in aqueoussolutions, which limits the quantity of drug that can bemicroencapsulated by usual techniques. In an effort to increase theamount of drug delivered to the target tissues, crystalline drugsuspensions are sometimes encapsulated. Fragile liposome or non-lipidcarriers too often rupture or are pierced by the sharp crystals,however, leading to loss of the drug before it reaches its target. Thisundesired release of the drug crystals has also been known to damage thelining of blood vessels.

Others have endeavored to increase the amount of drug in a liposome byloading the drug into the liposome by via a pH gradient. U.S. Pat. No.5,192,549 (issued to Barenolz and Haran) describes methods for formingliposomes and then obtaining transmembrane loading of amphiphatic drugsinto the liposomes using an ammonium ion gradient between the internaland external aqueous phase on either side of the liposome membrane. Themovement of ammonium from inside the liposome to the outside causes a pHchange inside, thereby creating a driving force for the amphiphatic drugto be loaded or released through the membrane. Disadvantages of thismethod are that it requires the encapsulation of ammonium sulfate oranother ammonium salt inside the liposomes, and transmembrane transportis limited to weak amphiphatic compounds. This type of drugconcentrating method has not been used successfully to form encapsulatedcrystals, however. If this method were applied to protein crystal growthinside the liposome, it would be limited to applications where theprotein was compatible with the ammonium salts and dissolved NH₄.

Another area where protein crystals are used is in macromolecularcrystallography, which requires large, high-quality protein crystals.Conventional methods of growing protein crystals, as required for x-raydiffraction studies of three-dimensional structure, are oftencompromised by the formation of multiple small crystals, amorphousprecipitates and aggregates rather than a single, or a few, largecrystals from the limited amount of protein in the available motherliquor. It has been estimated that about 10¹⁵ molecules are required tomake up a crystal of sufficient size for x-ray crystallographicexamination (Proteins Structures and Molecular Properties, 2nd Ed.,Thomas E. Creighton, Ed., W. H. Freeman and Co., NY, N.Y., p. 203). Itis often observed that, with conventional techniques, the best crystalsbegin to redissolve because of fluid perturbations at the crystalsurface, temperature shifts and other changes in the mother liquorsurrounding the crystal. Carrier fluids used to wash the crystal freefrom the mother liquor or used during mounting of the crystal (for x-raydiffraction) also tend to cause redissolution of the crystal before itcan be analysed.

There are many existing methods aimed at enhancing protein crystalgrowth, some of which take advantage of the favorable crystal growingconditions found in microgravity. An apparatus for carrying outcrystallization of proteins and chemical syntheses by liquid-liquiddiffusion in microgravity is described in U.S. Pat. No. 4,909,933(issued to Carter et al.) Another apparatus, disclosed in U.S. Pat. No.5,130,105 (issued to Carter et al.) relies on vapor diffusion growth ofprotein crystals. Other recent microgravity-dependent methods aredisclosed in U.S. Pat. No. 5,106,592 (issued to Stapelmann et al.),which deal with hanging drop vapor diffusion, dialysis of the proteinsolution, and interface diffusion between the protein solution and aprecipitating agent.

A ground-based (i.e., Earth normal gravity) method of concentratingprotein solutions to obtain crystal growth is described by Todd et al.((1990) J. Crystal Growth 110: 283-292), and U.S. Pat. No. 5,104,478(issued to S. K. Sikdar et al.), which relies on osmotic dewatering ofprotein solutions. Todd et al. and Sikdar et al. describe the use of adual chamber device wherein a near-saturated protein solution isseparated from a highly osmotic solution by a reverse osmosis membranewhich allows dewatering, resulting in supersaturated conditions which inturn cause nucleation and protein crystal growth in the mother liquor.The main advantage of this method is that the rate of dewatering can bedetermined by the difference in osmotic pressure on either side of themembrane. One drawback of this method is that the nucleation andsubsequent protein crystal growth depends on increasing theconcentration of precipitant and protein in the mother liquor. There isno control over the effects of solute driven convection on the surfaceof the crystal. As is the case with the protein crystals grown underconditions of microgravity, the crystals are not protected by anyenclosure thus they are subject to physical damage as they are harvestedand mounted. None of the existing methods for growing large, perfectcrystals provide adequately protected protein crystals.

In conventional x-ray diffraction studies to elucidate thethree-dimensional structure of a protein, in order to avoid physicaldamage to protein crystals, the crystals have typically been mounted inaqueous gels. There are problems, however, in removing the gel materialwithout affecting the integrity of the protein crystal. It would bedesirable if a protein crystal could be encapsulated in a shell ormembrane that was able to protect the crystal from harsh environmentswhich can cause degradation. A crystal contained within a closed,non-degrading environment would be useful to those working in fieldsrequiring high quality, intact protein crystals. Also needed is a way togrow larger and better quality protein crystals by eliminating some ofthe physical factors which perturb crystal growth and by bettercontrolling the dewatering conditions to promote single crystal growth.It would be desirable to have a method of preparing protein crystalsentrapped in liquid filled microcapsules surrounded by a thin, flexibleouter membrane, yet are sturdy enough to protect the enclosed crystalsfrom conditions which might cause fracture or fluid convection that canalter the molecular arrangement at the crystal surface, or dissolution.

Also needed are better carriers for drugs, particularly crystallinedrugs, which can resist prematurely rupturing and can provide sustainedand/or controlled release at a therapeutic target site, and protecttissues from the sharp edges of the crystals.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to providemicrocapsules containing solutions and/or crystals of bioactivesubstances such as drugs and proteins, that have semi-permeablemembranes which are rugged enough to protect fragile crystals and toresist shear and other mechanical forces typically associated withhandling of such crystals.

It is another object of the present invention to provide microcapsulescontaining highly ordered structures of other bioactive agents, orbiomolecules, such as DNA, RNA or oligonucleotides, which are capable ofbeing transported intact through the human vascular system for releaseat a desired site of action.

It is another object of the invention to provide microcapsules havingouter “skins” or membranes which avoid being readily detected andeliminated by the reticuloendothelial system, and which protect themicrocapsules against shear forces encountered during use, particularlyduring transport within the vascular system en route to target tissues.

Another object is to optimize the concentration of a bioactive agent ina microcapsule in order to achieve subsequent sustained or controlledrelease of the agent.

It is a further object of the invention to provide microcapsules whichprovide a closed environment that is favorable for growth of crystalsunder prescribed conditions of dewatering.

Still another object of the invention disclosed herein is to provide amethod for making custom microcapsules containing protein crystals ofsuitable quality for X-ray diffraction studies of native and activatedprotein structures.

A further object of the invention to provide larger microcapsularpackages containing saturated or near-saturated solutions of thesebioactive substances than has been possible before, and to provide anenvironment for these microcapsules that is conducive to growing largecrystals inside the microcapsule, or to accommodate larger 3-D orderedstructures than has previously been possible.

By entrapping protein crystals in these special purpose microcapsules,they can be protected from conditions which might cause fracture of thecrystals or fluid convection which can alter the molecular arrangementat the crystal surface, or dissolution. The semi-permeable membraneprovided by the invention not only protects the crystals from harshenvironments which can cause degradation, it also provides a closedenvironment which favors crystal growth under prescribed conditions ofcontrolled dewatering.

In addition to crystallizable proteins or drug that are chemicalcompounds, many other bioactive substances which are capable of forminga highly ordered structure may be similarly microencapsulated. Forinstance, a duplex DNA strand or RNA-like structure, or a concentratedsolution of an oligonucleotide or a polyribo- or -deoxyribonucleotide orother labile biological are also suitable for entrapment according tothe methods of the present invention.

Accordingly, the present invention provides a basic method of making amicrocapsule comprising preparing a first phase containing a firstsolvent, a co-solvent and a first polymer dissolved therein. The methodincludes preparing a second phase of different density than the firstphase, the second phase also including a second solvent, a surfactant, asalt, and a bioactive substance, all dissolved in the second phase. Inthis method the first polymer and surfactant are selected such that thehydrophobic/lipophilic balance value (HLB) of the surfactant is greaterthan the HLB of the first polymer. Thusly made, the first and secondphases are capable of forming a mutual interface.

The basic method of making a microcapsule that contains a protein orbioactive agent preferably also has a second polymer dissolved in thesecond phase. In this case, the first polymer, second polymer andsurfactant are each selected such that their respectivehydrophobic/lipophilic balance values (HLB) are in the following order:surfactant>second polymer>first polymer. Upon bringing the two phasestogether gently, to form an interface, and by limiting fluid shearforces at the interface to about 0-50 dynes/cm², microcapsulescontaining the dissolved bioactive agent are formed. Preferably theshear forces are limited to about 0-100 dynes/cm² so as to form largermicrocapsules.

In preferred embodiments of the invention, the bioactive substance is aprotein which is dissolved in the second phase at a concentration thatis at or near saturation. In some embodiments, a crystal of the proteinis also suspended in the second phase solution. If the protein isparticularly susceptible to degradation, a protein stabilizing agent maybe included in the second phase solution.

The first solvent is preferably water, methanol, ethanol, isopropanol,n-hexanol, or n-heptanol, or a hydrocarbon having a low or medium HLB5-10. The co-solvent is preferably a 3-carbon to 8-carbon (C₃-C₈) normalalcohol, tetrahydrofuran, dioxane, acetonitrile, dimethylformamide,dimethylacetamide, dimethylsulfoxide or a similar solvent.

The first polymer is preferably a polymer of glycerol monostearate,glycerol monooleate, glycerol monolaurate, glycerol dioleate, glyceroldistearate or other hydrophobic mono- or polyglycerides or waxy polymersof low molecular weight, or it can be a combination of any of thosepolymers. In an alternative method of the invention, however, the firstsolvent is water and the first polymer is a polyethylene glycol having amolecular weight greater than about 400 kd, cyclodextrin,polyvinylpyrrolidine or polyvinyl alcohol. In another alternativeembodiment, an alternative membrane forming material comprising a sterolor a phospholipid is substituted for the first polymer. The sterol orphospholipid may be cholesterol, stigmasterol, phytosterol, campesterol,phosphatydyl choline or CENTROLEX-F™.

In preferred methods of making the microcapsules of the presentinvention, the second solvent is water and the surfactant has ahydrophilic/lipophilic balance value (HLB) of about 10-40. The HLB ofthe first polymer is preferably less than the HLB of the surfactant by 2or more HLB units. The surfactant may be chosen from the groupconsisting of sorbitan monooleate plus ethylene oxide, dextran,polyethylene glycol (PEG), C₁₂-C₂₀ fatty acids, and quaternary NH₄salts.

The second polymer is preferably capable of adhering to the firstpolymer and is chosen from the group consisting of PEG 400-20000,dextran 4000-20,000, a polysaccharide of mol. wt. ranging from about10,000-100,000, polyvinylpyrrolidone (PVP), a polyvinyl alcohol andother similar polymeric materials.

In preferred embodiments the salt contained in the second phase solutionis NaCl, KCl, CaCl₂, quaternary NH₄ salts, cetyl trimethylammoniumbromide, 2-amino-2-methyl aminomethyl propanol or a similar salt.

According to the preferred methods of the invention, after initialformation of the microcapsule, the membrane is allowed to cure. Aftercuring, the relatively sturdy microcapsules may be separated intofractions of a certain size range, if desired for a particular purpose,such as injection into a blood vessel for therapeutic treatment. Aftercuring, the microcapsules may then be subjected to gradual dewatering inorder to gently bring about supersaturation of the bioactive agent andto encourage single crystal nucleation and growth. Optionally, anadditional coating of polymer may be applied to the microcapsule, aftercuring, after dewatering, or after full growth of the crystal has beenaccomplished, in order to provide a thicker, more protective skin on themicrocapsule.

The dewatering step of certain embodiments of the invention may includeexposing the microcapsule to a closed local environment which is capableof regulating the rate and extent of microcapsule dewatering wherebycontrolled crystallization of a protein occurs within said microcapsule.The dewatering step may include exposing microcapsules to a dewateringsolution containing a salt or a polymer which is excluded by thesemi-permeable membrane of the microcapsule. In an alternativeembodiment, the method includes diffusing a low molecular weight saltinto said interior cavity to induce single crystal nucleation andcrystal growth.

In certain embodiments employing a closed local environment fordewatering the microcapsule, the environment may also permit controllingthe protein concentration and the concentration of charged precipitantmolecules at or near the surface of a growing protein crystal so thatthe internal order and extent of crystallization of said protein crystalis optimized.

One preferred method of making a microcapsule includes preparing a firstphase containing a first solvent chosen from the group consisting of:methanol, ethanol, isopropanol, m-hexanol, or n-heptanol, a co-solventchosen from the group consisting of: a 3-carbon to 8-carbon (C₃-C₈)normal alcohol, and a first polymer dissolved in the first phase. Thefirst polymer is a hydrophobic mono- or polyglyceride. According to thismethod, a second phase is also prepared, the second phase having adifferent density than that of the first phase. The second phase iswater containing polyethylene glycol, as a surfactant, and a secondpolymer dissolved therein. This second polymer, which is capable ofadhering to the first polymer, is PEG 1000-8000. A protein is alsodissolved to saturation or near-saturation in the second phase.Optionally, one or more crystals may also be suspended in the secondphase solution. The second phase also includes NaCl dissolved therein.An important feature of this method is that the first polymer, secondpolymer and surfactant are chosen such that the hydrophobic/lipophilicbalance values (HLB) are: surfactant>second polymer>first polymer. Whenthe two phases are gently brought into direct contact, an interfaceforms between them. It is a critical part of this method that the fluidshear stress at the interface be limited to 0-100 dynes/cm² so thatmicrocapsules having the desired characteristics will form. Aftermicrocapsules have formed, the outer membrane is then cured to make itmore rugged and durable. Optionally, an additional polymer coating maybe applied over the outer membrane if an even thicker membrane, or skin,is desired.

Also provided in accordance with the present invention is an improvedmethod of determining the three-dimensional structure of a predeterminedprotein molecule by x-ray crystallography. The improvement includesforming a microcapsule containing a saturated or near saturated aqueoussolution of a protein surrounded by a semi-permeable polymeric membrane.The microcapsule is exposed to a dewatering solution having a higherosmotic pressure than the encapsulated protein solution, whereby wateris osmotically removed from said encapsulated protein solution. Bycontrolling the concentration of a dewatering agent in the dewateringsolution, gradual, ordered crystallization of the protein occurs withinthe microcapsule. This gradual, ordered crystal growth is allowed tocontinue until the crystal becomes at least about 50-300 microns acrossone face. A microcapsule containing a crystal of sufficient size andcrystalline quality is then carefully selected. The microcapsule ismounted in an x-ray capillary tube and subjected to a high energy x-raycrystallographic procedure to obtain a characteristic x-ray diffractionpattern of the protein crystal.

In an alternative and preferred method of performing x-raycrystallography on a crystal specimen, the present invention provides animprovement over methods which include isolating a crystal specimen in afiber loop with an attached handle portion, freezing the crystalspecimen, mounting the crystal specimen and fiber loop on a goniometerhead such that said crystal is positioned in a continuous N₂ streamloop, and rotating the goniometer head in an x-ray beam. The presentimprovement includes substituting for the conventional crystal specimena microencapsulated crystal that has a protective outer membranesurrounding a large crystal and a small amount of mother liquor. Themembrane, which is preferably a composite of two or more polymers, issubstantially transparent to the x-ray beam so that it does notinterfere with the x-ray diffraction pattern of the crystal. Accordingto this improved method, the membrane has an electrostatic charge whichrenders the microencapsulated crystal electrostatically attracted to thefiber loop. This electrostatic attraction is strong enough to supportthe microencapsulated crystal inside said loop. Optionally, a drop ofliquid may be adhered to the outer membrane of the microencapsulatedcrystal to facilitate freezing. In certain embodiments the membrane isnegatively charged and the loop is a fiber having a positiveelectrostatic charge. In certain preferred embodiments the crystal is ahighly ordered protein crystal.

The present invention also provides a microencapsulated protein crystalprepared by certain methods described above. In some embodiments themicrocapsule is best characterized as a product of a particular methodof the invention, because the inventors believe that there are as vetunrealized features and characteristics of the new microcapsules whichare attributable to the novel method of making.

In accordance with the present invention, a microcapsule is providedhaving an outer membrane surrounding an interior cavity, the interiorcavity containing a saturated or nearly saturated solution of abioactive agent. An important feature of this new microcapsule is thatit is capable of withstanding shear forces at least as great as theturbulent blood flow within a human artery.

Certain embodiments of the new microcapsule have a membrane containingat least one of the membrane forming material materials described abovein the summary of the methods, descriptions. In the preferredembodiments, the membrane is a composite containing a first polymer anda second polymer that is capable of adhering to the first polymer. TheHLB of the second polymer is preferably greater than the HLB of thefirst polymer.

In certain embodiments of the microcapsule of the present invention, theinterior cavity also contains the protein or bioactive agent in the formof a highly ordered structure such as a crystal. In some embodiments thecrystal substantially fills the interior cavity. The membrane may evensubstantially conform to the shape of a large crystal. In preferredembodiments, the membrane is resistant to rupturing or piercing by thecrystal.

In certain embodiments the microcapsule's membrane is permeable to waterand low molecular weight salts but impermeable, or only slightlypermeable, to the bioactive agent. In some embodiments, the membrane isless than or equal to 1 micron in thickness, and in others the membraneis about 3-5 microns thick.

Preferably the bioactive agent is a protein or a drug, however in someembodiments of the microcapsule of the invention the bioactive agent isa biomolecule such as a polypeptide, oligonucleotide, RNA, DNA or othercompound which can be crystallized.

Certain embodiments of the new microcapsule include a highly orderedstructure, such as a crystal, about 50-2000 microns in size.

Certain alternative embodiments of the microcapsule of the inventionhave an interior cavity that contains a hydrophobic phase surrounded byand partially immiscible with a saturated or near-saturated solution ofthe bioactive agent.

Also provided by the present invention is a composition comprising amultiplicity of certain microcapsule of the invention suspended in anaqueous solution having higher osmotic pressure than that of thebioactive agent solution. The higher osmotic aqueous solution mayinclude a dewatering agent capable of causing water to be transportedthrough said membrane and out of said interior cavity. This dewateringagent may be a salt or a high molecular weight polymer which is excludedby said membrane.

Certain embodiments of the new microcapsules have a polymeric membranethat is transparent to x-ray radiation and/or does not interfere withthe x-ray diffraction pattern of the highly ordered structure.

The present invention accordingly provides an x-ray crystallographyreagent for use in elucidating the three-dimensional structure of apredetermined biomolecule which is capable of forming a highly orderedstructure. The reagent comprises a dewatered microcapsule preparedaccording to certain methods of the invention and having a highlyordered structure, such as a protein crystal, substantially filling theinterior cavity of the microcapsule.

The present invention also provides a pharmaceutical compositioncomprising a pharmacologically effective multiplicity of certainmicrocapsules of the invention, together with a pharmacologicallyacceptable carrier. For particular medical uses, the average size of themicrocapsules is about 1-20 microns, and for others the average size ofsaid microcapsules is about 50-300 microns, or even greater than about300 microns.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a conceptual diagram of the procedure for forming amicrocapsule of the present invention and for obtaining crystal growthwithin the microcapsule and mounting the encapsulated crystal for x-raydiffraction analysis.

FIG. 1B shows conceptually a composite polymeric skin on certainmicrocapsules of the present invention.

FIG. 1C shows an alternative embodiment of the microcapsules of theinvention as used for x-ray crystallography.

FIG. 2 is a photomicrograph of lysozyme crystals grown inside amicrocapsule, formed under microgravity conditions, and having ahydrophobic, semi-permeable polymeric outer membrane in accordance withthe present invention (magnification=400×).

FIG. 3 show concanavalin A inside microcapsules of the presentinvention.

FIG. 3A is a photomicrograph showing concanavalin A crystals andsolution contained in a microcapsule of the present invention formed at1 g (magnification=150×).

FIG. 3B is a photomicrograph showing concanavalin A crystals andsolution contained in a microcapsule of the present invention formed inmicrogravity conditions aboard the Space Shuttle (magnification=150×).

FIG. 4 is a photomicrograph of amoxicillin crystals inside amicrocapsule of the present invention formed at 1 g.

FIG. 5 is a photomicrograph of a preformed lysozyme crystal that wassubsequently microencapsulated (1 g) and dewatered according to a methodof the present invention, and then submersed in water(magnification=100×).

FIG. 6 shows cis-platin crystals inside microcapsules of the presentinvention.

FIG. 6A is a photomicrograph of a single cis-platin crystal inside amicrocapsule of the present invention containing;

FIG. 6B is similar to FIG. 6A and shows the additional oil layer.

FIG. 6C is similar to FIG. 6A but includes superimposed fitted lines formeasurement of crystal size.

FIG. 7 is a conceptual drawing of an alternative embodiment of amicrocapsule of the present invention, having a membrane surrounding anaqueous solution containing a bioactive material which itself surroundsa partially immiscible hydrocarbon core.

FIG. 8A is a graph showing the distribution of sizes of microcapsulescontaining photofrin produced by a representative method of the presentinvention under low shear Earth normal gravity conditions.

FIG. 8B is a graph showing the distribution of sizes of microcapsulescontaining photofrin produced by a representative method of the presentinvention under low shear microgravity conditions.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Microgravity research has provided a new understanding of mechanisms offluid mechanics, interfacial behavior, and many biological processingmethods that are compromised by gravity-dependent phenomena. Asdemonstrated by the inventors in U.S. patent application Ser. No.08/349,169 (now U.S. Pat. No. 5,827,531), liquid microcapsules havingtwo or more concentric layers can be made in a single step by slowlybringing two immiscible liquid phases (of differing densities) togetherunder conditions where the surface tension and diffusive forces aregreater than the convective forces. The mechanisms of spontaneousformation of large microcapsules in microgravity have been furtherinvestigated by the inventors to develop new methods to make these andother unique microcapsules in both Earth-based and in microgravityenvironments. The disclosure of U.S. patent application Ser. No.08/349,169 (now U.S. Pat. No. 5,827,531) is incorporated herein byreference.

There are no prior art methods wherein protein crystals are encapsulatedin a semi-permeable membrane which protects the crystals from harshenvironments which can cause degradation. For the purposes of thepresent disclosure, the term “semi-permeable membrane” includes theusual definition and, when the context allows, also means that water andlow-molecular weight salts can pass through the membrane, but themembrane is impermeable to a protein or other bioactive agent containedby the microcapsule of the present invention.

Neither are there any prior art methods that provide a closedenvironment that favors crystal growth under prescribed conditions ofcontrolled dewatering within a microcapsule or inside a polymermembrane. Known methods utilizing osmotic dewatering for growing proteincrystals has relied on use of small chambers having a planar reverseosmosis membrane positioned between the mother liquor and the dewatering(high osmotic pressure) salt solution. The present method uses sphericalmicrocapsules wherein the entire outer membrane surface is available forosmotic dewatering, also for infiltration by hydrogen or hydroxyl ionsthereby changing the pH within the microcapsule to favor or enhanceprotein saturation and subsequent crystal growth. The membrane also canallow diffusion of salt ions into the microcapsule to decrease thesolubility of the protein or bioactive agent. The increased surface areaof the spherical microcapsule allows for more rapid change in conditionsthroughout the sphere of mother liquor, hence faster controlled changesall around the crystals which enhances the formation of more ordered andperfectly formed crystals.

EXAMPLE 1 General Procedure for Microcapsule Preparation

Gravity-dependent restrictions in the basic liquid-liquid spontaneousmicroencapsulation process led to the design of several microgravityexperiments to explore the utility of this process when density-drivenphenomena were eliminated. In particular, density-driven,gravity-dependent restrictions of the liquid-liquid microencapsulationprocess were: early phase separation producing fragile microcapsules;and interfacial dynamic flow causing coalescence of microcapsules.Failure of early ground-based experiments to derive uniformmicrocapsules lead to a desire to attempt microcapsule formation inspace.

The microgravity flight experiments led to the development of aliquid-liquid microencapsulation process that involves use ofsurfactants and co-surfactants in the aqueous phase andco-solvent/co-surfactant alcohols in the organic phase, which alsocontained high molecular weight polymers that formed a tough, yetflexible, outer “skin” on the final microcapsules. The inventors'earlier work in microgravity conditions included a single stepdispersion which produced unique multi-lamellar microcapsules containingvarious aqueous drugs co-encapsulated with iodinated poppy seed oil (aradiocontrast medium with a sp. gravity=1.35). Subsequent ground controlexperiments also produced some of these unique microcapsules andillustrated that the Earth normal (1 g) process could be improved toyield useable microcapsules by varying the liquid phase formulations.

In further studies, it has become clear that the flexible outer skins,or membranes substantially improve the ruggedness of the microcapsulesformed. The inventors have gone on to develop a method of formingmicrocapsules that have a low molecular weight, water permeable outer“skin” or membrane surrounding a sphere of aqueous mother liquorcontaining a dissolved protein. The method utilizes aprotein-impermeable polymer dissolved in a liquid organic phase.Formation of the microcapsule occurs by interfacial coacervation at theimmiscible interface of the organic and aqueous phases, trapping theprotein “mother liquor” inside.

The general procedure for forming microcapsules is essentially asdisclosed by the inventors in U.S. Pat. No. 5,827,531, and as describedbelow. The disclosure of that application is incorporated herein byreference, to the extent that it provides details supplementary to thoseset forth herein.

The method of the present invention relies on liquid-liquidinteractions. In the basic method, the first step entails formulating afirst phase or layer (“Phase 1”) while the second step entailsformulating a second phase or layer (“Phase 2”). The two phases areformulated to be substantially immiscible with one another. For thepurposes of this invention, “immiscible” means that the two adjoiningphases or layers have sufficiently different densities, viscosities orsurface tensions to permit the formation of a mutual interfaceresembling a meniscus, or visible interface.

As shown in Table 2 and the details that follow, formulating Phase 1comprises combining a first solvent (which is preferably hydrocarbonbased), a first polymer soluble in the first phase and a co-solvent. Thefirst polymer is selected to be one which is soluble in the first phaseand when formed into the microcapsule membrane is permeable to aqueoussolvents and ions but impermeable to proteins or peptides. A smallamount of a co-solvent is also added to the first phase, whichco-solvent may also function as a co-surfactant.

The method next calls for formulating a second phase (“Phase 2”), whichis preferably aqueous and is immiscible with the first phase. The secondphase comprises a second solvent, a second polymer soluble in the secondphase but insoluble in the first phase, a surface active agent(“surfactant”), a dissolved salt and a dissolved protein or otherbioactive substance of interest. The bioactive substance may also be inthe form preformed crystals suspended in a concentrated solution of thesame bioactive substance.

In order to ensure that the liquid-liquid interactions necessary to formthe microcapsule will occur, certain of the constituents of Phases 1 and2 are selected relative to one another (i.e., based on certaincharacteristics of one component, the second is selected advantageouslybased on its respective characteristics. After selecting the immiscibleorganic and aqueous solvents, the surface active agent in the secondphase is selected such that it will have a hydrophilic/lipophilicbalance (“HLB”) value greater than that of the first polymer constituentof the first phase (Polymer 1). Generally, the most useful surfaceactive agents are those which are nonionic and which have ahydrophilic/lipophilic balance value of 10.0 or greater. Next, thesecond polymer constituent of the second phase (Polymer 2) is selectedto have a hydrophilic/lipophilic balance value lower than that of thesurface active agent constituent of the same phase. While not anexhaustive list, certain hydrophilic/lipophilic balance values ofmaterials which may be used in the formulations of the invention asPolymer 1 or Polymer 2 are provided in Table 1 below. See McCutcheon'sDetergents and Emulsifiers (1979) North American Edition, McCutcheonDivision, MC Publishing Co., 175 Rock Road, Glen Rock, N.J. 07452, forspecific HLB values ranging from 2 to 42, see pages 23-39; and for HLBvalues ranging from 0.5 to 30.5, see pages 228-241. In making suitablepolymer selections, it is important that the first polymer have an HLBvalue less than that of the surface active agent which, in turn, has anHLB value which is greater than that of the second polymer.

TABLE 1 HYDROPHILIC/LIPOPHILIC BALANCE (HLB) (McCutcheon 1979) CompoundHLB Glycerol treioleate 0.8 Cholesterol 1.0 Triglyceride of coconut oil1.4 Sorbitan trioleate 1.8 Sorbitan tristearate 2.1 Glycerol monooleate2.7 Mono and di glycerides of fat-burning fatty acids 2.8 GlycerolMonostearate (gms) 2.8-5.0 (3.8 preferred) Propoxylated ethylene diamineplus 3-28 ethylene oxide Mono/diglyceride 3.2 Glycerol mono coconut 3.4Mono/diglyceride 3.5 Propylene glycol mono fatty acid ester 3.5Monoethoxyl lauryl ether 3.6 Stearyl lactyl acid 3.8 Hydrogenatedcottonseed oil 3.8 Sodium lauryl sulfate 4.0 Mono and diglycerides withcitric acid or 4.2-4.6 lactylic ester or fatty acid Ethoxylated fattyamine (2 moles ETO) 4.5 Diethylene glycol monostearate 4.7 Sorbitanmonopalmitate 4.7 Diethylene glycol monostearate and oleate 4.7Ethoxylated (2) cetyl ether 5.3 Glycerol Monoricinoleate 6.4 Glycerolmonolaurate 6.8 Triglycerol mono stearate 7.0 Polyethylene glycol (400dioleate) 7.2 Lanolin sterol 8.0 Ethoxylated nonyl phenol (CO-420 & CO850) 8.0-16.0 Polyethylene glycol (400) distearate 8.2 Sorbitanmonolaurate 8.6 Ethoxylated sorbitan fatty acid esters 9.0 andalkyl/aryl alcohol Anhydrous lanolin 10.0 Polyethylene glycolmonostearate 11.0 Polyethylene glycol 400 11.2 Ethoxylated (10) cetylether 12.9 Ethoxylated glycerol monostearate (GMS) 13.1 Sorbitanmonostearate 14.9 Sorbitan monooleate with 20 moles ethylene 15.0 oxideEthoxylated (20) oleyl ether 15.3 Ethoxylated (20) stearyl cetyl ether15.8 Ethoxylated castor oil 18.0 Nonyl phenol polyethylene glycol ether18.1 Polyethylene glycol 600 mono laurate 19.6 Sodium lauryl sulfate 40Propylene glycol monostearate 40 Hydroxylated lanolin sodium oleylsulfate 42 Blends of GMS and sorbitan monooleate 52 with 20 molsethylene oxide

TABLE 2 General Formulations for the Two Phases Phase 1 Phase 2 75-90%(v/v) Solvent 1 70-98% (v/v) Water  0-20% (v/v) Co-solvent  1-4% (w/v)Surfactant  1-5% (w/v) Polymer 1  1-10% (w/v) Polymer 2  1-3% (w/v) Salt 1% to saturation Bioactive Substance

Solvent 1

Solvent 1 is preferably ethanol, methanol, isopropanol, n-hexanol, orn-heptanol, or another hydrocarbon having a low or medium HLB of 5-10.As a less preferred alternative, water may be substituted for thehydrocarbon if, for example, it is desirable to avoid the use of ahydrocarbon.

Co-Solvent

The Co-solvent is a 3-carbon to 8-carbon (C₃-C₈) normal alcohol,tetrahydrofuran, dioxane, acetonitrile, dimethylformamide,dimethylacetamide, dimethylsulfoxide or the like. An acceptableco-solvent also acts as a surfactant and preferably has a highdielectric constant, i.e. in the range of about 10-20.

Polymer 1

Suitable outer skin forming compounds which can be used as Polymer 1are: glycerol monostearate, glycerol monooleate, glycerol monolaurate,glycerol dioleate, glycerol distearate, or other hydrophobic mono- orpolyglycerides or waxy polymers of low molecular weight, or acombination of the foregoing polymers, which are capable of forming athin, semi-permeable membrane.

Alternatively, a hydrophobic aqueous phase may be substituted for thePhase 1 liquid, if desired, as mentioned above with respect toSolvent 1. In this alternative water is used as the primary solvent anda polymer such as a high molecular weight polyethylene glycol (mol. wt.greater than 400 kd), cyclodextrin, polyvinylpyrrolidine or polyvinylalcohol is dissolved therein. The particular polymer selected should beinsoluble in the second aqueous phase (Phase 2).

It was somewhat surprising to the inventors that other chemicals notstrictly considered polymers, such as lecithins, when used to make themicrocapsules of the invention, also laid down a polymer-like skin orfilm at the interfaces of the two phases. Thus, depending on thecharacteristics of the particular bioactive agent selected forencapsulation, the following sterols and phospholipids may besubstituted for Polymer 1: cholesterol, the plant sterols stigmasterol,phytosterol and campesterol, or lecithins such as phosphatydyl choline(CENTROLEX-F™). In any event, acceptable hydrocarbon-soluble polymers(or non-polymeric membrane forming materials which can substitute forPolymer 1), will generally have lower HLB values, in the order of lessthan 1 to about 12. Polymer 1 is insoluble or sparingly soluble inwater.

Polymer 2

Polymer 2 is water soluble and is preferably chosen from the following:PEG 400-20000, dextran 4000-20,000, a polysaccharide of mol. wt. rangingfrom about 4,000-100,000, polyvinylpyrrolidone (PVP), a polyvinylalcohols or another similar polymeric material. Polymer 1 and Polymer 2are selected such that they cannot solubilize each other, and they donot chemically react to form new and distinct adducts. PreferablyPolymer 2 is capable of adhering to Polymer 1, however, and in preferredformulations the microcapsule's final, or “cured,” membrane is actuallya composite of Polymers 1 and 2. In this case, Polymer 2 serves toenhance the strength of the outer membrane or skin, which is primarilymade up of Polymer 1. It should be noted that the inventors havesucceeded in making protein- and crystal-filled microcapsules using onlyPolymer 1 as the skin-forming material. However, a sturdier membrane isobtained when the second polymer, Polymer 2, is included. Using the samerationale for selection, additional suitable polymers could be includedin the Phase 1 of Phase 2 solutions to form the composite polymericskin.

Surfactant

A suitable surfactant for dissolving in the aqueous Phase 2 solution canbe any of the following ionic or non-ionic compounds: sorbitanmonooleate plus ethylene oxide, dextran, polyethylene glycol (PEG),C₁₂-C₂₀ fatty acids or a quaternary NH₄ salt. The surfactant chosen willtypically have a higher HLB value, in the range of about 10 to 40. Thesurface active agent is selected so that it has an HLB valuesufficiently different (preferably HLB>2 or more units different) fromthat of either polymer 1 or 2, so that the surfactant will lower theinterfacial tension just enough to promote film (outer skin) formationby polymer 1 and/or polymer 1 plus polymer 2. but does not lower thesurface tension enough to cause complete emulsification of the skinpolymer(s) in the second solvent. The surfactant itself does not form amajor component of the outer film, or skin, which surrounds themicrocapsule, encapsulating the drug or other bioactive agent.

Salt

Suitable salts are NaCl, KCl, CaCl₂, quaternary NH₄ salts, cetyltrimethylammonium bromide, 2-methyl-2amino-aminomethyl propanol oranother similar salt.

Bioactive Substance

The protein or bioactive substance, or agent, is preferably a proteinthat can be crystallized, but it may also be another bioactive substanceor agent that is compatible with the microcapsule-forming materials andis capable of forming a crystal or other highly ordered structure. Someother types of molecules that are suitable for encapsulating arestructures such as a double stranded DNA α-helix, polypeptides,oligonucleotides, D- or L-stereoisomers, pharmaceutical compounds, toxicagents, and the like. A protein stabilizing agent may also be includedin the Phase 2 solution when the substance to be microencapsulated is aprotein that is easily degraded. Some suitable protein stabilizingagents are benzamidine and 2-MP.

Optionally, an oil can be added to the Phase 1 solution. For example, insome applications it may be desirable to include a marker or tracer fortracking the location of the microcapsule in the body. A radiocontrastmaterial such as iodinated poppy seed oil (IPO) could be incorporatedinto the microcapsule to permit detection by radiographic techniques.

The preceding formulations have been used successfully by the inventorsin both Earth normal and in microgravity environments to formliquid-filled microcapsules.

The basic method next creates an interface between the first and secondphases. The creation of the interface is achieved in such a way thatminimal shear and mixing occurs between the phases. The two immisciblephases are brought together in such a mechanical manner that the fluidshear properties are controlled to low levels from 0 to 100 dynes/cm²,preferably below 50 dynes/cm², and most preferably 12 dynes/cm² orbelow, such that the adsorptive surface properties at the immiscibleinterfaces are not significantly altered. Although the exact mechanismsare not fully understood, the inventors believe that the maintenance ofcertain adsorptive surface properties, such as the surface tension,Helmholtz charge distribution (electrical double layer), andpartitioning of the surfactant molecules between the immiscible phasesmust remain substantially intact so that lateral phase separation ofphases can occur to form the water/organic interface. This is believedto be the mechanism for the formation of multi-lamellar vesicles whichare formed in a single step, as discussed in U.S. patent applicationSer. No. 08/349,169 (now U.S. Pat. No. 5,827,531). Although bestdemonstrated under microgravity conditions, wherein buoyant convectionis absent and diffusion-driven convection predominates, favorablemicrocapsule forming conditions are also accomplished in unit gravityenvironment by balancing the density differences between the two liquidphases or by any other mechanical means which prevents excess fluidshear from significantly altering the normal adsorptive surfaceproperties which are determined by the chemical composition of theformulas and the interfacial phenomena among the solvents, polymers andsurfactants, as described above. One way of creating a suitableinterface is by sliding individually separated compartments containingthe two phases into register with one another in a manner thatsubstantially limits shear and provides gentle mixing, as shown in theschematic illustration in FIG. 1A.

Interface Formation

The two liquid phases formulated as described above are placed intoseparate compartments or spaces which are each connected to a centraldiffusion chamber into which each compartment can deliver its residentliquid loading. The compartments are initially closed to access into thecentral diffusion chamber so that the first and second liquids are keptapart from one another and not allowed to interact. The separation ofthe two liquids is maintained until both liquids and the devicecontaining them can be placed in an environment in which convectivemixing may be minimized, such as in a microgravity environment. FIG. 1Ais a schematic illustration of one way that a liquid-liquid interfacemay be achieved, and shows conceptually the structure of themicrocapsules formed. While it is possible to use any number of devicesto accomplish this separation, a preferred apparatus for use in creatingthe optimal low-shear conditions favoring spontaneous interfacialcoacervation is the Materials Dispersion Apparatus (MDA) (manufacturedby ITA, Inc., Exton, Pa.), which is described in U.S. patent applicationSer. No. 08/349,169 (now U.S. Pat. No. 5 827,531). Another suitableapparatus is the Microencapsulation Electrostatic Processing System(MEPS), which is described in a companion U.S. patent applicationentitled “Microencapsulation and Electrostatic Processing Device”application Ser. No. 09/079,833(NASA No. MSC-22937-1-SB), whichapplication is incorporated in pertinant part herein by reference. Anysuitable apparatus capable of slowly and gently bringing two immiscibleliquid phases of differing densities together and permitting spontaneousformation of microcapsules may be used however, as long as the requiredlow shear conditions (i.e., 0-100 dynes/cm²) are maintained. Shearforces of 35 dynes/cm² and below have been demonstrated by the inventorsduring the spontaneous formation of representative microcapsules at theinterface of the two phases under conditions of Earth normal gravity andin microgravity, as illustrated in FIGS. 3A and 3B, for example.

Calculations were made for shear forces occurring in various types ofapparatus which have been used to successfully make the multi-layeredmicrocapsules. Original calculations for shear stress in cell syringeswere extended to calculations for Liquid Mixing Apparatus (LMAs) made byITA, Inc. in Exton, Pa. Microcapsules were formed using severalrepresentative formulations taken from Table 2, for the alcoholic(Phase 1) and aqueous (Phase 2) phases.

In order to form microcapsules, the interfacial surface tension must notbe overcome by shear stresses, at least until the “outer skin” hasformed and stabilized, after which the microcapsules can withstand evenhigher shear stress. The toughened outer skin can be observed byobserving a sample of the microcapsule suspension under the microscope.The microcapsules tumble in gentle fluid flow rather than disintegrate.A basic assumption is that the microcapsules do not form efficiently inthe high shear stress fields within a distance equal to three (3)diameters from the container walls or immiscible interface. Thefollowing calculations indicate that most 10 micron diametermicrocapsules would be formed in the boundary layer from 30 microns to0.2 cm of the immiscible interface.

Shear-Laminar Flow Boundary Layer calculations for formingrepresentative microcapsules including the following assumptions: thetwo fluids are incompressible, immiscible and one acts similar to asolid wall; the viscosity of Phase 1 (Med-10) at 20° C. (μ¹) is 0.01716g/cm-sec; the viscosity of Phase 2 (aqueous Cis-Platinum-II) (μ²) is0.0101 g/cm-sec; the average dynamic viscosity (μ′) is 0.01363; thedensity of Phase 1 (ρ¹) is 0.802; the density of Phase II (ρ²) is 1.009;the effective kinematic viscosity (ν′) is 0.015703, where(ν′)=((μ¹/ρ¹)+(μ²/ρ²)/2; the maximum fluid velocity along the containerwall or immiscible interface=V_(max), i.e., 14.29 cm/sec.

Shear Stress (τ) is$\tau = \frac{(0.332)\quad (\mu)\quad ( V_{\max} )\sqrt{\frac{V_{\max}}{\upsilon^{\prime}}}}{\sqrt{x}}$${{where}\quad \mu} = \frac{( {\mu^{1} + \mu^{2}} )}{2}$${{and}\quad \tau} = \frac{1.949812}{\sqrt{x}}$

where x=distance (cm) from wall or interface

Therefore, when x=

μm cm 30 0.003 50 0.005 100 0.010 200 0.020 300 0.030 500 0.050 6000.060 900 0.090 1000 0.100

then τ=

(dynes/cm²) 35.6 27.57 19.50 13.79 11.26 8.72 7.96 6.50 6.17

Thus, the fluid shear stress at 30 microns is 35.6 dynes/cm² and isreduced to 11 dynes per cm² at 300 microns from the interface. In eachinstance, microcapsules were observed to form. Experiments performed inthe inventors' laboratory indicate that the microcapsules of mostinterest (i.e. >10 microns in diameter) do not form within 3-4 diameters(30-50 microns) from the interface. The microcapsules described in theseExamples were formed using the basic procedure described above, in shearfields calculated to be approximately 35 dynes/cm². The largestmicrocapsules (i.e., ranging from about 50 microns to 2 mm), which areespecially preferred for crystal x-ray diffraction studies, are probablybest formed in the more quiescent region of the boundary layer which isabout 100 microns or farther from the container wall. This correspondsto a shear stress field of 19 dynes/cm² or less. When microcapsules ofabout 20 microns in size are desired, such as for in vivo use as drugcarriers, relatively higher shear conditions are permissible.

Minimal shear conditions are critical in gravity dependent conditions(i.e., ≧1 g). In this case, the optimum microcapsules are formed whenthe fluid shear between the immiscible Phase 1 and Phase 2 liquids isrestricted to 10-100 dynes/cm². Under microgravity conditions (i.e., <1g) the fluid shear forces can be better controlled in the range of about2-30 dynes/cm².

In the next step of the basic method, conditions are established forsubstantially limiting all mixing between the interfaced liquid phases.Preferably, the two phases are allowed to interact at their interfacewithout agitation, stirring, shearing or like force for a period ofabout 1-10 minutes. Preferably the temperature is maintained withinabout ±1° C. of the maximum solubility temperature of the polymer, butbelow the denaturation temperature of the protein or other bioactivesubstance. For representative proteins and drugs demonstrated in theseexamples, temperatures up to 43° C. are maintained. It is preferred tolimit even those quiescent forces such as gravity-controlledsedimenting, shifting, drift and the like. Thus, it is low fluid shear,chiefly diffusion-driven convection, that is used to spontaneously formmicrocapsules, as the chemical formulations of the different phasesassist in lowering the surface free energy across the interface. It isalso at this time that formation of the polymeric outer coating isinitiated. The inventors' recent investigations indicate that, in eitherunit gravity or microgravity conditions, it is advantageous, however,that the two phases be allowed to interact at their interface with asmall amount of fluid shear force applied (approximately 2-40dynes/cm²), in order to increase the yield of microcapsules. The maximumpractical shear force is a function of the kinematic viscosity of eachfluid at the interface. The shear force should not produce a Reynoldsnumber greater than 300. Uniformity and sphericity is a commoncharacteristic of the microcapsules of the invention, regardless of thegravity environment in which they are produced.

As shown in FIG. 1A, the microcapsules 10 have an aqueous solution 30 ofa bioactive substance 50 in the interior compartment 12 and havepolymeric outer coatings or skins. Polymer 1 and Polymer 2 together forma rugged composite polymer membrane or shell 65, after curing. Polymer 1primarily makes up the outermost part 60. FIG. 1B illustratesconceptually how the two polymers are thought to be distributed when themicrocapsule initially forms. Polymer 1 (60) and Polymer 2 (70) aresubstantially distinct layers, at least initially, with Polymer 2 (70)adhering to Polymer 1 from the inside of the microcapsule and providingenhancement of the outermost membrane formed by Polymer 1.

In the present studies, large, uniformly spherical microcapsules havebeen prepared without using conditions of microgravity for limitingmixing between the phases. Limitation of interactions between the phasesis best promoted by substantially balancing the specific gravity betweenthe phases. By varying the density of the polymer solutions, convectioncan be reduced, as further described below. In either microgravity orunit gravity, using the procedures and rationale described herein,mixing between the two phases is controlled such that it is chiefly theresult of diffusion-driven convection and not a function of densitydriven bouyancy, and fluid shear is limited to about 2-100 dynes/cm² andinterfacial mixing is counteracted by the surface free energy of therespective phases.

Curing of the Membrane

Studies on the Space Shuttle permitted 10 minute dispersion timesfollowed by curing of the outer skin (exemplified by a polyglyceride)for eight days under microgravity conditions. The microcapsules formedin the microgravity environment are generally obtained in better yieldand of larger average size than at Earth normal gravity, as shown inFIGS. 8A and 8B. At 1 g, a wide range of sizes generally smallermicrocapsules is typically obtained. The frequency vs. size data shownin FIGS. 8A and 8B were obtained for representative microcapsulescontaining the drug photofrin. These microcapsules contained photofrinin the form of a saturated or nearly-saturated solution variously withor without photofrin crystals. The inventors have observed that thenature of the active material that is encapsulated can affect theresulting size range of the microcapsules.

The distribution of microcapsules obtained by a representative procedureranges from uniform spheres of about 1 to 300 microns in diameter. Byvarying the formulations so as to reduce the density differentialbetween the aqueous and organic phases, even larger microcapsures areformed, up to about 2 mm, as previously discussed. The average size ofthe microcapsules formed in one representative procedure, shown in FIG.8B was about 35 microns.

The ground-based production of microcapsules is able to replicate thesize range (roughly 5-250 microns in a representative study), but theaverage size microcapsule is typically about 10-40 microns in diameter,as shown in FIG. 8A. At least a partial reason for this wider sizedistribution at Earth-normal gravity is the apparent inability at 1 g toavoid certain sedimentation phenomena alone and sedimentation effectscombined with weight-related contact of sedimented microcapsules. Thegravity-dependent deformations of the spherical microcapsules as theyform give rise to areas of thinner polymer deposition. Thus, theflexible microcapsules formed under microgravity conditions tend to havemore uniform size distributions than those formed in Earth-normalgravity, are more rugged, and have a higher average diameter thanground-made microcapsules, largely due to the absence of thermalconvection, buoyancy forces, and instabilities that occur at theimmiscible interfaces.

These factors necessitate some additional manipulation underEarth-normal environments that is not required in the microenvironment,namely, size separation of the resulting microcapsules in order toobtain fractions of more uniform size microcapsules. Therefore, at 1 gthe durability of the outer coating of the microcapsules of the presentinvention becomes even more important. The curing step significantlyenhances the ruggedness of the Earth-normal microcapsules. Anoutstanding characteristic of these microcapsules is their sturdiness,as demonstrated by their ability to withstand size segregation byfiltration or sieving.

The microcapsules of the present invention are equal or superior in sizeand function to microcapsules prepared by conventional methods. Theinventors believe that there are no previously known methods of makingmicrocapsules designed for growing a protein crystal inside themicrocapsule. Due to their novel methods of making, the microcapsulesprepared as described herein may have other significant features oradvantages over known microcapsules that cannot be fully identified orappreciated at the present time.

EXAMPLE 2 Protein Crystallization within a Microcapsule

Referring now to FIGS. 1A and 2, microcapsules containing therepresentative protein lysozyme were prepared essentially according tothe general method described in Example 1. The Phase 2 formulationcontained 7% w/v lysozyme from hen egg, and 0.01M sodium acetate, pH4.0, dissolved in water.

In FIG. 1A, the production of microcapsules is schematically shown.Microcapsule 10 contains the Phase 2 solution 30, containing lysozymemolecules 50 in the interior cavity or compartment 12. The microcapsuleinitially has an outer polymeric membrane 65, made up primarily ofPolymer 1, and an adherant polymeric layer 70 (FIG. 7B), which isprimarily Polymer 2. After letting the microcapsules rest quiescently inthe Phase 1 and Phase 2 solutions for up to about 1 hr., the outer skin65 had fully formed and stabilized (cured) and the microcapsules arethen able to withstand shear stresses equivalent to the fluid flow in ahuman artery, or a Reynolds number of up to about 300.

The microcapsule skin is preferably less than 1 micron thick whencrystal growth within the microcapsule is of primary interest, as in thepresent example. When the primary goal is to place a protective, lesspermeable coating around a preformed crystal or to provide a carrier fora concentrated protein solution, however, a thicker polymeric skin ofabout 2-3 microns is preferred. The thickness of the skin may beincreased, and the permeability decreased, by suspending themicrocapsules in the Phase 1 solution, or in a similar alcoholic/polymer1 solution, to apply an additional layer of polymer over the outer skin.Repeated applications will build up the skin to the preferred degree ofthickness. As part of this skin thickening step, a “handle” such as animmunoglobulin-bound polymer, may be included, if desired.

Optionally, a fractionating step such as filtration or sieving, may beperformed to isolate a fraction of microcapsules within a specified sizerange. The fluid surrounding the microcapsules was then exchanged for adewatering fluid 120 having a higher osmotic pressure such that watermolecules diffused out of the semi-permeable outer membrane. Thedewatering fluid contained 25% (w/v) NaCl dissolved in deionized water.Alternatively, another similar salt could be used, at a concentrationthat provides an osmotic value at least 10% greater than that of themother liquor. Similarly, a solution of 18% (w/v) higher molecularweight material such as polyethylene glycol (PEG) 8000 dissolved indeionized water may also be used for dewatering the microcapsules, oranother high molecular weight PEG (of about 4-20 kDa) may besubstituted.

Referring still to FIGS. 1A and 2, the higher osmotic pressure of thedewatering fluid 120 causes water molecules to diffuse out of thesemi-permeable outer membrane 65 of the microcapsules, therebydehydrating the mother liquor 35 to nucleate lysozyme crystals 55. Thedewatering fluid sustains osmotic conditions that favor the growth ofthe lysozyme crystals within the microcapsules. The dewatering processis continued until extensive dewatering of the microcapsule is achieved,after which only a small amount of the mother liquor 35 remains and theflexible skin of the microcapsule substantially conformed to the shapeof the large crystal. As can be seen in FIG. 2, the lysozyme crystalessentially fills the interior of the microcapsule. A lysozyme crystalproduced in this way is of high quality, due to its large size andinternal structural order. The protective skin that surrounds the motherliquor and the crystal permits the crystal to grow unperturbed bycontact with a container wall or with other crystals. After completionof crystal growth, the tough, somewhat elastic, outer skin continues toprotect fragile crystalline structures from breakage, and deterredpenetration by the sharp crystal edges. Preferably the outer skin isabout one micron or less in thickness, for obtaining optimum dewateringrates.

Microcapsules containing the lysozyme crystals are convenientlyharvested either by 1) suspending in a liquid carrier phase with amicropipette or 2) capturing with a hydrophobic fiber loop, preferablyone having a surface charge more or less opposite that of themicrocapsule. The microcapsules are then ready for analysis by polarizedlight microscope to determine the size and shape of the crystal(s)contained inside or mounting in an x-ray capillary tube, as illustratedin FIG. 1A. Because the crystal is bathed in mother liquor within themicrocapsule, drying out of the crystal in the x-ray capillary tube isretarded or eliminated. The polymeric membrane components selected forformulating the microcapsules are selected to be transparent to x-raysand are preferably non-x-ray diffracting. FIG. 1C shows an alternativeembodiment of the microcapsules of the invention as used for x-raycrystallography in a procedure that omits use of a quartz capillarytube. Use of the microcapsules for x-ray diffraction studies isdiscussed in more detail in Example 4, below.

This method provides distinct advantages over prior art methods ofgrowing protein crystals by providing a closed environment which favorscrystal growth under prescribed conditions of controlled dewatering. Itavoids the problem of having a large crystal fall out of a hanging drop,as frequently encountered in conventional vapor diffusion methods. Italso avoids having the crystal touch a container wall. In the past,typical methods employing osmotic dewatering for growing proteincrystals have relied on use of small chambers having a planar reverseosmosis membrane positioned between the mother liquor and the dewatering(high osmotic pressure) salt solution. The present method uses sphericalmicrocapsules wherein the entire outer membrane surface is available forosmotic dewatering. The spherical membrane also optimizes conditions forinfiltration by hydrogen or hydroxyl ions, thereby changing the pHwithin the microcapsule to favor or enhance protein saturation andsubsequent crystal growth. The increased surface area of the sphericalmicrocapsule allows for more rapid change in conditions throughout thesphere of mother liquor, hence faster controlled changes all around thecrystals which enhances the formation of more ordered and perfectlyformed crystals.

By varying the choice and relative amounts of the Phase 1 and Phase 2components and/or by fractionating the microcapsules, one can easilyoptimize the size range of the microcapsules needed for a particularprotein, drug compound, or other bioactive agent. For some uses, such asin vivo drug carriers, microcapsules of 5-25 microns will be preferred.For other uses, uniform spherical microcapsules of greater than 25microns are needed; and in still other applications such as x-raycrystallography specimens, very large 50-2000 micron crystals protectedby an inert membrane covering are wanted.

FIG. 3 shows another representative protein crystal, thephytohemagglutinin concanavalin A, which was encapsulated andcrystallized essentially as described for lysozyme. The aqueous phasecontained 40 mg/ml ConA, 4.25 g/l sodium nitrate, 47.4 mg/l manganesechloride, 27 mg/l calcium chloride, and 43.7 mg/l TRIS acetate buffer,pH 6.5, dissolved in water. FIG. 3A shows microcapsules containing ConAcrystals and mother liquor, as formed at Earth normal gravity. FIG. 3Bshows the same type of microcapsules formed under conditions ofmicrogravity on board the Space Shuttle.

The above-described procedure can be readily modified, if desired, bythe encapsulation of a first phase comprised of an aqueous,near-saturated solution of protein, or other bioactive substance, and acarefully selected hydrophobic surfactant which is capable of permeatingthrough the membrane such that the entrapped hydrophilic proteinmolecules become super-saturated. The super-saturated condition can becontrolled such that nucleation of one or a few, well-ordered crystalsoccurs as the surfactant is transported through the semi-permeable“outer membrane” of the microcapsules.

Another way to enhance protein crystal formation within themicrocapsules is to add a salt to the surrounding fluid As withconventional salting out procedures, any suitable non-denaturing saltsuch as (NH₄)₂SO₄ or NaCl can be used, provided that it can diffusethrough the polymeric skin to cause the gradual “salting out” of theprotein or it causes dewatering through the membrane.

In an alternative dewatering process, the basic method may be modifiedby encapsulating an aqueous near-saturated solution of protein in asemi-permeable membrane and then suspending the microcapsules in acarefully chosen dewatering fluid which, upon being subjected to acontrolled electrostatic field, regulates the dewatering rate from themicrocapsules by controlling the local salt concentrations on eitherside of the semi-permeable membrane. By controlling the concentration ofprotein and charged precipitant molecules at or near the surface of thegrowing protein crystal, the quality and size of the resulting crystalstructure can be further optimized.

A suitable method and an apparatus for applying an electrostatic fieldto the microcapsule are disclosed in the inventors' related applicationscross-referenced above. It is believed that no crystal growth-enhancingmethod in use today employs electrostatic fields to manipulate the localconcentration of protein molecules or charged precipitant molecules nearthe surface of the crystal as it is growing. Although there are numerousways of establishing an electrostatic field, one suitable apparatus isdescribed in the inventors' related application (cross-referenced above)describing the Microcapsule and Electrostatic Processing System (MEPS)for controlling precipitant and protein concentrations inside theprotective environment of the microcapsules. The disclosure of thatapplication is incorporated herein by reference, to the extent that itprovides details supplementary to those set forth herein.

The inventors have also made the surprising discovery that addition ofenergy in the form of uv light to the contents of the protein-containingmicrospheres also appears to cause a change in chemical equilibrium,producing supersaturation, crystal nucleation and acceleration ofcrystal growth. Ongoing investigations by the inventors are aimed atclarifying the mechanism by which this occurs.

The inventors believe that there are no previously known methods ofmaking microcapsules designed for growing a protein crystal in theinterior cavity of the microcapsule.

EXAMPLE 3 Encapsulation of a Preformed Protein Crystal and ContinuedCrystal Growth

FIG. 5 is a photomicrograph taken at about 100× magnification showing alarge lysozyme crystal that was encapsulated as a large preexistingcrystal suspended in the Phase 2 solution, prepared as described inExample 2 and saturated or nearly saturated with dissolved lysozyme. Themicrocapsule was subsequently submerged in water for 12 hours prior tobeing photographed. It can be readily seen that the large crystalsubstantially fills the interior cavity and the skin around themicrocapsule conforms to the shape of the crystal, with only a thinlayer of mother liquor remaining between the crystal faces and the skin.The permeability of the membrane was such that the lysozyme crystal hadredissolved only minimally over the 12 hour water exposure period. Thisestablishes that the speed of water diffusion through the membrane canbe limited to a very slow rate, for optimizing crystallizationconditions. If desired, the microcapsule can be submerged in thealcohol/polymer 1 solution to receive an additional coating of polymer.This would provide a thicker, less permeable, more rugged polymer coatfor the microcapsule, on the order of about 3-5 microns.

This demonstrates that it is possible to form sturdy membranes aroundexisting crystals of a representative protein and thereby provideprotection for even the most fragile crystals. The encapsulated crystalavoids the problem of redissolution of thin crystalline structures,typically encountered with most conventional crystal growing andhandling procedures.

The microcapsules formed by the processes described in Examples 1-3 havethe unique characteristic that they can be exposed to high osmoticsolutions of salts, polymers, etc. which will cause dewatering of themother liquor within the microcapsules, thereby maintaining apreexisting crystalline structure and even allowing continued crystalgrowth within the protective confines of the outer membrane. The presentexample also demonstrates that when the microcapsule has received athicker polymer coating the microcapsule offers even more protection tothe crystal, while reducing water permeability and redissolution of thecrystal.

EXAMPLE 4 Crystal Preparation for X-ray Diffraction Analysis

Microcapsules containing protein crystals measuring about 50-2000microns on a side, are prepared as described in Examples 1-3. ThePolymer 1 and Polymer 2 materials chosen for use are preferablytransparent to x-ray wavelengths, so as not to cause interferingdiffraction patterns. The particular formulations used are optimized forformation of very large microcapsules containing a saturated ornear-saturated protein solution by generating the microcapsules underconditions of very low shear field. Microcapsules containing crystals ofthe desired size are individually selected and carefully transferredinto an x-ray capillary tube or other mount so the crystal can beorientated in a high energy x-ray beam for diffraction studies, inaccordance with established methods. The encapsulated crystals aresufficiently protected by the skin of the microcapsule such that evenfragile, sharp-edged crystals can be manipulated successfully into thecapillary tubes.

Representative microcapsules containing large lysozyme crystals wereconveniently harvested either by 1) suspending in a liquid carrier phasewith a micropipette or 2) capturing with a hydrophobic fiber loop. Themicrocapsules were then ready for analysis by polarized light microscopeto determine the size and shape of the crystal(s) contained inside ormounting in an x-ray capillary tube, as illustrated in FIG. 1A. Becausethe crystal is bathed in mother liquor within the microcapsule, dryingout of the crystal in the x-ray capillary tube is retarded oreliminated.

As illustrated in FIG. 1C, alternatively, a microcapsule containing alarge, high quality crystal is also suitable for advantageous use in anx-ray crystallography procedure that avoids the problem of manipulatinga specimen into a quartz x-ray capillary tube. Such a method generallyincludes “lassoing” a crystal specimen, together with a drop of liquid,in a fiber loop. While the crystal is held in suspension within theloop, supported by the surface tension of the surrounding liquid, theloop is placed into liquid propane to freeze the crystal specimen. Theloop is then manipulated by its handle and the handle is mounted on thegoniometer head of the x-ray device. The frozen crystal, together withthe associated mother liquor, is thereby situated in a continuous streamof nitrogen gas so that the crystal remains frozen while beingmanipulated under the x-ray beam. Referring to FIG. 1C, a microcapsule10 containing a large crystal 55 of any desired substance, especially aprotein or drug crystal, prepared as described herein, is captured,supported and manipulated by a fiber loop 130 without relying on liquidsurface tension to hold the specimen in place. Not only does the sturdy,x-ray transparent membrane 65 of the microcapsule protect the fragilecrystal structure while the specimen is being “teased” into the fiberloop, the polymeric skin can readily be adjusted to have anelectrostatic charge on its surface. By advantageously choosing thefiber loop material and/or by applying an opposite electrostatic chargeto the fiber loop, the microencapsulated crystal specimen is held inplace in the loop and supported by electrostatic attraction. Forexample, the microcapsule membrane may have a negative charge and thepolymer fiber loop may be positively charged. Some suitable fibermaterials are nylon, cellulose and polyethylene terphthalate. Themicroencapsulated crystal specimen is then frozen and situated under thex-ray beam in the same manner as other crystals via handle 140 attachedto the goniometer head 150. The new microcapsules containing largecrystal specimens of 50-300 microns, and even up to about 2 mm in size,can be supported electrostatically and manipulated with the fiber loopfor examination by x-ray crystallography.

EXAMPLE 5 Protein Crystal Growth between Two Solvent Phases within aMicrocapsule

FIG. 7 is a conceptual drawing of a layered microcapsule madeessentially as described in Examples 1-3. An aqueous saturated or nearlysaturated solution of protein, or other bioactive agent (30) isencapsulated, between a non-aqueous, hydrocarbon phase (20) which isonly partially miscible with the aqueous solution (30) and a third,solid phase composed of a semi-permeable polymer that forms the outermembrane (65) and which is in contact with a high osmotic concentrationaqueous solution of salts or polymers (120). Dewatering from the proteinsolution (30) occurs by migration of water into the hydrocarbon liquidphase (20) or by transport out of through the semi-permeable membraneinto the high osmotic solution (120). The particular hydrocarboncomponents selected for the core liquid, or hydrocarbon phase (20) arechosen based on their characteristics of being slightly miscible withwater to the desired degree. This embodiment of microcapsules may beadvantageous for more rapidly dewatering certain proteins or otherbioactive agents.

EXAMPLE 6 Encapsulation and Crystallization of Cis-Platin

Referring now to FIGS. 6A and 6B, a 2% (w/v) solution of therepresentative pharmaceutical drug cis-platinum was microencapsulatedessentially as described in Example 1, however 5% (w/v) iodinated poppyseed oil (IPO) was also included in the Phase 1 solution. Theformulation is described in Table 3.

TABLE 3 Phase 1 (“MED 10”) Phase 2 88.0% isopropyl alcohol 0.2%Cis-platinum  2.5% n-hexanol 1% PEG-4000  2.5% n-heptanol 5% Dextran-40(MW = 40,000)  5.0% IPO 1% Sorbitan Monooleate-20 moles ethylene oxide 2.0% H₂O 0.8% NaCl  5% w/w Glycerol Monosterin Balance - sterile waterfor injection (GMS)

This concentration of cis-platinum was sufficient to allow nascentcrystal formation within the microcapsule. In this case, crystalformation occurred at or near the time of formation of the microcapsulecontaining the dissolved pharmaceutical material. It can readily beappreciated that the components of the aqueous solvent system used todissolve an aqueous-soluble pharmaceutical agent may be advantageouslyselected, and their concentrations adjusted, to permit water moleculesto migrate away from the drug-containing layer into the alcoholicmixture. The process of crystal formation is likely to be promoted inthis manner after formation of the microcapsule. The results obtained inthis investigation for cis-platinum is considered to be representativeof other bioactive agents which can be similarly encapsulated.

Using the above-described methods, an aqueous solution of a drug orother bioactive agent which is non-saturated may be brought tosuper-saturation and crystal nucleation after encapsulation, viacontrolled transport of water out of the microcapsule's interiorcompartment, as described above. In this way it is possible to enhancethe crystallization process after the microcapsule is formed. As shownin FIGS. 2-4, the crystal thus formed may take up most of the internalcapacity of the microcapsule, i.e. about 65-90% of the internal volume.

Another representative drug, the antibiotic amoxicillin, wasencapsulated essentially as described above, and the resultingmicrocapsule containing amoxicillin crystals and mother liquor is shownin FIG. 4.

If desired, co-encapsulation of a radio-contrast medium, as shown inFIG. 6B, enables oncologists to monitor the delivery of anti-tumormicrocapsules to target tumors using computerized tomography andradiography that track the distribution of microcapsules after releasefrom the intra-arterial catheter. Such microcapsules will have importantapplications in chemotherapy of certain liver, kidney, brain and othertumors.

The diameters of microcapsules possible to attain using the methods ofthe invention are also of particular usefulness in medical applications.Thus, whereas prior art methods have been able to routinely producemicrospheres of about 1-10 micron average sizes, the present methodsprovide similarly-sized microcapsules of 1-20 micron diameters forintravenous administration. Also provided are 50-300 micron sizedmicrocapsules particularly useful in interarterial chemoembolization oftumors, and microcapsules in the range of 300 micron and greaterdiameters useful in interperitoneal administered drugs. The membrane orskin around the outer surface of the microcapsule avoids being readilydetected and largely eliminated by the reticuloendothelial system (RES).The outer skin protects the microcapsules against shear forcesencountered during manufacturing processes and during transport withinthe vascular system enroute to the target tissues. The hydrophobic outermembrane can also be modified, by selection of advantageous polymericand/or non-polymeric components, so as to retard oxygen transport andthereby reduce oxidative degradation of the entrapped drug. This wouldlikely improve the shelf-life of parenteral suspensions. The flexible,deformable outer skin on the microcapsules of the invention providesincreased packing densities within vascular beds. This results inmicrocapsules superior to prior art solid microspheres (e.g. gelatin,albumin or starch) commonly used for chemoembolization therapy againsttumors. It is expected that the new microcapsules, carrying moreconcentrated amounts of drugs which are already known to betherapeutically effective, will be even more effective as substitutesfor existing liposome encapsulated drugs. The thin, semi-permeablemembrane of some embodiments of the new microcapsules permits controlledrelease of the encapsulated drug at the desired site of action. Sincethe concentration of drug in a microcapsule and its release rate areeasily determined, the correct microcapsule dosage for a particular drugcan be calculated from the customary dosages for that drug.

As another option, the polymeric skin may be made initially insoluble inPhase 2 but slowly solubilizable in physiological body fluids (e.g.,blood, serum, plasma, extracellular fluid, saliva, mucus).Alternatively, sustained or controlled release at a therapeutic targetsite may be obtained by modifying the above-described method to providea somewhat thinner, drug-semi-permeable membrane on the microcapsule.Upon injecting into a person in need of the drug, the microcapsulesserve to protect tissues, arterioles and veins from the sharp edges ofthe drug crystals.

While the preferred embodiment of the invention has been shown anddescribed, modifications thereof can be made by one skilled in the artwithout departing from the spirit and teachings of the invention. Forexample, a saturated non-aqueous solution of a drug could also besimilarly encapsulated and then exposed to conditions which would removesolvent through the outer membrane, producing a similar supersaturatedcondition, crystal nucleation and/or acceleration of the drug crystalgrowth, within a microcapsule. The embodiments described herein areexemplary only, and are not limiting. Many variations and modificationsof the invention disclosed herein are possible and are within the scopeof the invention. Accordingly, the scope of protection is not limited bythe description set out above, but is only limited by the claims whichfollow, that scope including all equivalents of the subject matter ofthe claims.

REFERENCES

Allen, T. M., Mehra, T., Hansen, C. and Chin, Y. C., “Stealth Liposomes:An Improved Sustained Release System for1-b-D-Arabinofuranosylcytosine,” Cancer Res. 52:2431-39, 1992.

Barenolz, Y. and Haran, G., “Method of Amphiphatic Drug Loading inLiposomes by pH Gradient,” U.S. Pat. No. 5,192,549, issued Mar. 9, 1993.

Carter, Daniel C., “Apparatus for Mixing Solutions in Low GravityEnvironments,” U.S. Pat. No. 4,909,933, issued Mar. 20, 1990.

Carter, Daniel C., “Protein Crystal Growth Tray Assay,” U.S. Pat. No.5,130,105, issued Jul. 14, 1992.

Martin, et al. “Microreservoir Liposome Composition and Method,” U.S.Pat. No. 5,225,212, issued Jul. 6, 1993.

Todd, P., Sidkar, S. K., Walker, C. and Korszun, Z. R., “Application ofOsmotic Dewatering to the Controlled Crystallization of BiologicalMacromolecules and Organic Compounds,” J. Crystal Growth 110: 283-292(1990).

Willoughby et al., “Neutral Glycolipid as an Adsorbent for Enteric ViralPathogens,” U.S. Pat. No. 5,192,551, issued Mar. 9, 1993.

Woodle, et al. “Liposomes with Enhanced Circulation Time,” U.S. Pat. No.5,013,556, issued May 7, 1991.

What is claimed is:
 1. An improved method of determining thethree-dimensional structure of a predetermined protein molecule by x-raycrystallography including mounting a crystal in an x-ray capillary tubeand subjecting said crystal to a high energy x-ray crystallographicprocedure to obtain a characteristic x-ray diffraction pattern of saidprotein crystal, wherein the improvement comprises: forming amicrocapsule containing a saturated or near saturated aqueous solutionof said protein surrounded by a semi-permeable polymeric membrane;exposing said microcapsule to a dewatering solution having a higherosmotic pressure than said encapsulated protein solution whereby wateris osmotically removed from said encapsulated protein solution;controlling the concentration of a dewatering agent in said dewateringsolution such that gradual, ordered crystallization of said proteinoccurs within said microcapsule; growing said crystal to at least about50-300 microns; harvesting the resulting crystal-containingmicrocapsule; selecting a microcapsule containing a crystal ofsufficient size and crystalline quality for obtaining an x-raydiffraction pattern.
 2. An improved method of determining thethree-dimensional structure of a predetermined protein molecule by x-raycrystallography including isolating a crystal specimen of said proteinin a fiber loop, freezing said crystal specimen, mounting said crystalspecimen and fiber loop on a goniometer head such that said crystal ispositioned in a continuous cold N₂ stream loop and kept frozen, androtating said goniometer in an x-ray beam, wherein the improvementcomprises substituting for said crystal specimen a microencapsulatedcrystal comprising a protective outer membrane surrounding an interiorcavity, said interior cavity containing a saturated or nearly saturatedsolution of a protein and containing a crystal of said proteinsubstantially filling said interior cavity, said membrane beingtransparent to the x-ray beam.
 3. The method of claim 2 wherein saidmembrane comprises an electrostatic charge such that saidmicroencapsulated crystal is electrostatically attracted to said loop,said electrostatic attraction being sufficient to support saidmicroencapsulated crystal inside said loop.
 4. The method of claim 2wherein a drop of liquid is adhered to the outer membrane of saidmicroencapsulated crystal.
 5. The method of claim 2 wherein the outermembrane of said microcapsule is negatively charged and said loopcomprises a fiber having a positive electrostatic charge.
 6. The methodof claim 2, wherein said fiber is nylon, cellulose or polyethyleneterphthlate.
 7. The method of claim 2, wherein said crystal of saidprotein is a highly ordered structure.
 8. The method of claim 2, whereinsaid fiber has a surface charge opposite that of said microcapsule.